Genome-Wide Association Study and Admixture Mapping Reveal New Loci Associated with Total Ige Levels in Latinos

Henry Ford Health System Henry Ford Health System Scholarly Commons

Center for Health Policy and Health Services Center for Health Policy and Health Services Research Articles Research

6-1-2015

Genome-wide association study and admixture mapping reveal new loci associated with total IgE levels in Latinos

Maria Pino-Yanes

Christopher R. Gignoux

Joshua M. Galanter

Albert M. Levin Henry Ford Health System, [email protected]

Catarina D. Campbell

See next page for additional authors

Follow this and additional works at: https://scholarlycommons.henryford.com/chphsr_articles

Recommended Citation Pino-Yanes M, Gignoux CR, Galanter JM, Levin AM, Campbell CD, Eng C, Huntsman S, Nishimura KK, Gourraud P, Mohajeri K, O'Roak B, Hu D, Mathias RA, Nguyen EA, Roth LA, Padhukasahasram B, Moreno- Estrada A, Sandoval K, Winkler CA, Lurmann F, Davis A, Farber HJ, Meade K, Avila PC, Serebrisky D, Chapela R, Ford JG, Lenoir MA, Thyne SM, Brigino-Buenaventura E, Borrell LN, Rodriguez-Cintron W, Sen S, Kumar R, Rodriguez-Santana JR, Bustamante CD, Martinez FD, Raby BA, Weiss ST, Nicolae DL, Ober C, Meyers DA, Bleecker ER, Mack SJ, Hernandez RD, Eichler EE, Barnes KC, Williams KL, Torgerson DG, Burchard EG. Genome-wide association study and admixture mapping reveal new loci associated with total IgE levels in Latinos. Journal of Allergy and Clinical Immunology 2015; 135(6):1502-1510.

This Article is brought to you for free and open access by the Center for Health Policy and Health Services Research at Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Center for Health Policy and Health Services Research Articles by an authorized administrator of Henry Ford Health System Scholarly Commons. Authors Maria Pino-Yanes, Christopher R. Gignoux, Joshua M. Galanter, Albert M. Levin, Catarina D. Campbell, Celeste Eng, Scott Huntsman, Katherine K. Nishimura, Pierre-Antoine Gourraud, Kiana Mohajeri, Brian O'Roak, Donglei Hu, Rasika A. Mathias, Elizabeth A. Nguyen, Lindsey A. Roth, Badri Padhukasahasram, Andres Moreno-Estrada, Karla Sandoval, Cheryl A. Winkler, Fred Lurmann, Adam Davis, Harold J. Farber, Kelley Meade, Pedro C. Avila, Denise Serebrisky, Rocio Chapela, Jean G. Ford, Michael A. Lenoir, Shannon M. Thyne, Emerita Brigino-Buenaventura, Luisa N. Borrell, William Rodriguez-Cintron, Saunak Sen, Rajesh Kumar, Jose R. Rodriguez-Santana, Carlos D. Bustamante, Fernando D. Martinez, Benjamin A. Raby, Scott T. Weiss, Dan L. Nicolae, Carole Ober, Deborah A. Meyers, Eugene R. Bleecker, Steven J. Mack, Ryan D. Hernandez, Evan E. Eichler, Kathleen C. Barnes, Keoki L. Williams, Dara G. Torgerson, and Esteban G. Burchard

This article is available at Henry Ford Health System Scholarly Commons: https://scholarlycommons.henryford.com/ chphsr_articles/135 Genome-wide association study and admixture mapping reveal new loci associated with total IgE levels in Latinos

Maria Pino-Yanes, PhD,a,b Christopher R. Gignoux, PhD,a,c,d Joshua M. Galanter, MD, MAS,a,c Albert M. Levin, PhD,e Catarina D. Campbell, PhD,f Celeste Eng, BS,a Scott Huntsman, MS,a Katherine K. Nishimura, MHP,a Pierre-Antoine Gourraud, PhD,g Kiana Mohajeri, BS,f Brian J. O’Roak, PhD,f,h Donglei Hu, PhD,a Rasika A. Mathias, PhD,i Elizabeth A. Nguyen, BS,a Lindsey A. Roth, MA,a Badri Padhukasahasram, PhD,j Andres Moreno-Estrada, MD, PhD,d Karla Sandoval, MS, PhD,d Cheryl A. Winkler, PhD,k Fred Lurmann, MS,l Adam Davis, MA,m Harold J. Farber, MD, MSPH,n Kelley Meade, MD,m Pedro C. Avila, MD,o Denise Serebrisky, MD,p Rocio Chapela, MD,q Jean G. Ford, MD,r Michael A. Lenoir, MD,s Shannon M. Thyne, MD,t Emerita Brigino-Buenaventura, MD,u Luisa N. Borrell, DDS, PhD,v William Rodriguez-Cintron, MD,w Saunak Sen, PhD,x Rajesh Kumar, MD, MSPH,y Jose R. Rodriguez-Santana, MD,z Carlos D. Bustamante, MA, PhD,d Fernando D. Martinez, MD,aa,bb Benjamin A. Raby, MD, MPH,cc Scott T. Weiss, MD,cc Dan L. Nicolae, PhD,dd Carole Ober, PhD,dd Deborah A. Meyers, PhD,ee Eugene R. Bleecker, MD,ee Steven J. Mack, PhD,ff Ryan D. Hernandez, PhD,c Evan E. Eichler, PhD,f,gg Kathleen C. Barnes, PhD,i L. Keoki Williams, MD, PhD,j,hh Dara G. Torgerson, PhD,a and Esteban G. Burchard, MD, MPHa,c San Francisco, Palo Alto, Petaluma, Oakland, and Vallejo, Calif, Madrid, Spain, Detroit, Mich, Seattle, Wash, Portland, Ore, Baltimore and Frederick, Md, Houston, Tex, Chicago, Ill, Bronx, NY, Mexico City, Mexico, San Juan, Puerto Rico, Tucson, Ariz, Boston, Mass, and Winston-Salem, NC

Background: IgE is a key mediator of allergic inflammation, and its levels are frequently increased in patients with allergic Abbreviations used disorders. eQTL: Expression quantitative trait locus Objective: We sought to identify genetic variants associated GALA I: Genetics of Asthma in Latino Americans with IgE levels in Latinos. GALA II: Genes-environments & Admixture in Latino Americans GPHB5 Methods: We performed a genome-wide association study and : Glycoprotein hormone beta 5 GRAAD: Genetic Research on Asthma in the African Diaspora admixture mapping of total IgE levels in 3334 Latinos from the GWAS: Genome-wide association study Genes-environments & Admixture in Latino Americans (GALA II) Indel: Insertion-deletion study. Replication was evaluated in 454 Latinos, 1564 European KCNH5: Potassium voltage-gated channel, subfamily H Americans, and 3187 African Americans from independent studies. (eag-related), member 5 gene Results: We confirmed associations of 6 genes identified by means RHOJ: Ras homolog family member J gene of previous genome-wide association studies and identified a novel SNP: Single nucleotide polymorphism genome-wide significant association of a polymorphism in the zinc ZNF365: Zinc finger protein 365 gene finger protein 365 gene (ZNF365) with total IgE levels (rs200076616, P 5 2.3 3 1028). We next identified 4 admixture mapping peaks (6p21.32-p22.1, 13p22-31, 14q23.2, and 22q13.1) at which local African, European, and/or Native American ancestry was significantly associated with IgE levels. The most significant peak was 6p21.32-p22.1, where Native American ancestry was associated with lower IgE levels (P 5 4.95 3 1028). All but 22q13.1 IgE is a class of antibody involved in host defense mechanisms against parasitic infections.1,2 Increased total IgE levels are were replicated in an independent sample of Latinos, and 2 of the 1 3 peaks were replicated in African Americans (6p21.32-p22.1 and strongly associated with allergic disorders and asthma severity. Monoclonal antibodies against IgE have proved to be effective in 14q23.2). Fine mapping of 6p21.32-p22.1 identified 6 genome-wide 4,5 significant single nucleotide polymorphisms in Latinos, 2 of which decreasing asthma exacerbations and allergic inflammation, replicated in European Americans. Another single nucleotide highlighting the importance of IgE in the cause of both asthma and allergic disease. polymorphism was peak-wide significant within 14q23.2 in 6-8 African Americans (rs1741099, P 5 3.7 3 1026) and replicated in The high heritability of IgE levels (47% to 80%), the clinical non–African American samples (P 5 .011). and biological relationship of IgE with atopic diseases, and the ability to measure IgE levels in serum with a high precision make IgE a Conclusion: We confirmed genetic associations at 6 genes and 1,9 identified novel associations within ZNF365, HLA-DQA1, and useful endophenotype for the study of atopic diseases. Conse- quently, many linkage analyses and candidate gene association 14q23.2. Our results highlight the importance of studying 9 diverse multiethnic populations to uncover novel loci studies have identified genes associated with IgE levels. Recently, associated with total IgE levels. (J Allergy Clin Immunol 5 genome-wide association studies (GWASs) have revealed single 2015;135:1502-10.) nucleotide polymorphisms (SNPs) near 12 genes to be associated with IgE levels at a genome-wide significance level, including 6 Key words: IgE, genome-wide association study, admixture genes within the major MHC.10-13 mapping, allergy, asthma, next-generation sequencing, Latinos, Only 1% of subjects within prior GWASs were Latinos,10-14 Hispanics, minority populations and therefore studying diverse populations might uncover some

1502

From the Departments of aMedicine, cBioengineering and Therapeutic Sciences, and Affymetrix. C. R. Gignoux has received research support from the University of Cal- xEpidemiology and Biostatistics, University of California, San Francisco; bCIBER ifornia San Francisco and the National Institutes of Health (MIH; T32 GM007175) and de Enfermedades Respiratorias, Instituto de Salud Carlos III, Madrid; dthe Department has stock/stock options in 23andme.com. A. M. Levin has received research support of Genetics, Stanford University, Palo Alto; ethe Department of Public Health Sci- from the National Institutes of Health (NIH) and is employed by the Henry Ford Health ences, jthe Center for Health Policy and Health Services Research, and hhthe Depart- System. C. D. Campbell has received research support from the American Asthma ment of Internal Medicine, Henry Ford Health System, Detroit; fthe Department of Foundation. K. K. Nishimura, R. A. Mathias, P. C. Avila, S. Sen, J. R. Rodriguez- Genome Sciences, University of Washington, Seattle; gthe Department of Neurology, Santana, D. L. Nicolae, and C. Ober have received research support from the NIH. University of California; hMolecular & Medical Genetics Department, Oregon Health P.-A. Gourraud is a board member for Methodomics SARL; has received research sup- and Science University, Portland; ithe Division of Allergy & Clinical Immunology, port from the Patient-Centered Outcomes Research Institute National Institutes of Department of Medicine, Johns Hopkins University, Baltimore; kthe Basic Research Health Hilton Foundation, the National Science Foundation, and the Office of the Laboratory, Center for Cancer Research, National Cancer Institute, Leidos Biomed- Navy; and has patents for personalized medicine for multiple sclerosis. C. A. Winkler ical, Frederick National Laboratory for Cancer Research, Frederick; lSonoma Tech- has received research support from the National Cancer Institute and the NIH (contract nology, Petaluma; mChildren’s Hospital and Research Center Oakland; nthe HHSN26120080001E). F. Lurmann has received research support from the National Department of Pediatrics, Section of Pulmonology, Baylor College of Medicine and Institute of Environmental Health Sciences (NIEHS, ES015794). H. J. Farber has Texas Children’s Hospital, Houston; othe Department of Medicine and yChildren’s received research support from the NIH/NIEHS; is employed by the Texas Children’s Memorial Hospital and the Feinberg School of Medicine, Northwestern University, Health Plan; has provided expert testimony for Serpe, Jones, Calendar, and Bell; and Chicago; pthe Pediatric Pulmonary Division, Jacobi Medical Center, Bronx; qInstituto has received payment for lectures and travel support from the American Academy of Nacional de Enfermedades Respiratorias (INER), Mexico City; rthe Department of Pediatrics. J. G. Ford has consultant arrangements with GlaxoSmithKline. E. Brigino- Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore; sBay Buenaventura has received research support, consulting fees, travel support, and pro- Area Pediatrics, Oakland; tthe Department of Pediatrics, University of California vision of writing assistance, medicines, equipment, or administrative support from the San Francisco, San Francisco General Hospital; uthe Department of Allergy and Sandler Foundation through the University of California San Francisco. R. Kumar has Immunology, Kaiser Permanente–Vallejo Medical Center, Vallejo; vthe Department received research support from the National Heart, Lung, and Blood Institute (NHLBI, of Health Sciences, Graduate Program in Public Health, City University of New HL093023) and the NIG. C. D. Bustamante is a member of the Scientific Advisory York, Bronx; wVeterans Caribbean Health Care System, San Juan; zCentro de Neu- Boards for Personalis and Ancestry.com and is a member of the Medical Advisory mologıa Pediatrica, San Juan; aathe Arizona Respiratory Center and bbthe BIO5 Insti- Board for Invitae; has consultant arrangements with 23andme.com, Etalon.com, and tute, University of Arizona, Tucson; ccthe Channing Division of Network Medicine, National Geographic; has received research support from the NIH; has a patent through Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Stanford University for DNA capture technology; and receives royalties from Person- Boston; ddthe Department of Human Genetics, University of Chicago, Chicago; eethe alis. F. D. Martinez has received research support from the NIH and has received pay- Center for Genomics and Personalized Medicine Research, Wake Forest School of ment for lectures from Abbott and Merck. D. A. Meyers and E. R. Bleecker have Medicine, Winston-Salem; ffChildren’s Hospital Oakland Research Institute, Oakland; received research support from the NHLBI (HL101651). S. J. Mack has consultant ar- and ggthe Howard Hughes Medical Institute, Seattle. rangements with Stanford University; is employed by Children’s Hospital and Supported by grants from National Institutes of Health: the National Heart, Lung and Research Center Oakland; has received research support from the NIH, National Insti- Blood Institute (HL088133, HL078885, HL004464, HL104608, and HL117004 to tute of Allergy and Infectious Disease, and National Institute of General Medical Sci- E.G.B; HL101651 to C.O.; HL093023 to R.K.; HL075419, HL65899, HL083069, ences; has received travel support from the National Marrow Donor Program; and has HL066289, HL087680, HL101543, and HL101651 to S.T.W.; and HL111636 to received payment from the American Society of Histocompatibility and Immunoge- J.M.G.); the National Institute of Environmental Health Sciences (ES015794 to netics. E. E. Eichler has received research support from the American Asthma Foun- E.G.B.); the National Institute on Minority Health and Health Disparities dation (AAF #10-0159); has served on scientific advisory boards for DNA Nexus, (MD006902 to E.G.B.); the National Institute of General Medical Sciences Pacific Biosystems, and SynapDx; and has consultant arrangements with Kuning Uni- (GM007546 to E.G.B. and J.M.G and GM007175 to C.R.G.); the National Institute versity of Science and Technology (KUST). K. C. Barnes has received research sup- of Allergy and Infection Diseases (AI079139 and AI061774 to L.K.W); the National port from the NIH and the NHLBI; is a member of the AAAAI/ACAAI Joint Board Cancer Institute (contract HHSN26120080001E by the Intramural Research Program Review Course; is employed by Johns Hopkins University; has received payment of the NIH, National Cancer Institute, Center for Cancer Research to C.A.W.); General for lectures from Northwestern University; and has received royalties from UpToDate. Clinical Research Centers Program (M01-RR00188 to the Texas Children’s Hospital L. K. Williams has received research support from the National Institute of Allergy and General Clinical Research Center); the National Center for Advancing Translational Infectious Disease, the NHLBI, and the NIH. E. G. Burchard has received research Sciences (KL2TR000143 to J.M.G.). Other sources of funding included Fundacion support from the NIH (HL088133, HL078885, HL004464, HL104608, and Ramon Areces (to M.P.Y.); the American Asthma Foundation (to E.E.E., L.K.W., HL117004), the National Institute on Minority Health and Health Disparities and E.G.B.); an RWJF Amos Medical Faculty Development Award (to E.G.B.); the (MD006902), and the American Asthma Foundation. The rest of the authors declare Sandler Foundation (to E.G.B.); the Mary Beryl Patch Turnbull Scholar Program (to that they have no relevant conflicts of interest. K.C.B.); an Ernest S. Bazley Grant (to P.C.A); UCSF Chancellor’s Research and Received for publication February 4, 2014; revised September 6, 2014; accepted for pub- Dissertation Year Fellowships (to C.R.G.); the Hewett Fellowship (to J.M.G); and lication October 15, 2014. the Howard Hughes Medical Institute (to E.E.E). The content of this publication is Available online December 6, 2014. solely the responsibility of the authors and does not necessarily reflect the views or pol- Corresponding author: Maria Pino-Yanes, PhD, Department of Medicine, University of icies of the Department of Health and Human Services, nor does mention of trade California, San Francisco, 1550 4th St, Rock Hall Rm 582, UCSF Box 2911, names, commercial products, or organizations imply endorsement by the US San Francisco, CA 94143-2911. E-mail: [email protected]. Government. 0091-6749/$36.00 Ó Disclosure of potential conflict of interest: M. Pino-Yanes has received research support 2014 American Academy of Allergy, Asthma & Immunology from Fundacion Ramon Areces and has received payment for lectures from http://dx.doi.org/10.1016/j.jaci.2014.10.033

case-control study of asthma in Latino children (Puerto Rican, Mexican, and (1041), we established a Bonferroni P value threshold for significance as other Latinos) recruited from the mainland United States and Puerto Rico and follows: approved by all local institutional review boards. All participants/parents pro- 25 vided written assent/consent, respectively. a 5 0:05 1041 5 4:8310 ; Total serum IgE levels were measured in duplicate on the ImmunoCAP 100 33 system (Phadia, Kalamazoo, Mich). Subjects were genotyped on the Axiom which is similar to Shriner et al. Statistically significant admixture mapping LAT1 array (World Array 4; Affymetrix, Santa Clara, Calif),25 as described peaks were brought forth for replication in the GALA I study and the African elsewhere.23 American EVE Consortium studies by using linear regression models. The Genetics of Asthma in Latino Americans A linear mixed-effects model was used for one study with extended pedigree structure (GRAAD-AC).34 A meta-analysis was performed with METASOFT (GALA I) study. Characteristics of the 454 asthma cases from the 35 GALA I study used for replication are shown in Table E1. Subjects were re- to assess the overall effect size of local ancestry across studies. cruited from the United States and Mexico City. Total IgE levels were Fine mapping of significant admixture mapping peaks was performed by measured, as described in the GALA II study, and genotyping was performed using allelic association testing with both genotyped and imputed SNPs. on the Affymetrix 6.0 GeneChip (Affymetrix).22 A Bonferroni correction based on the number of genotyped SNPs within each peak was used to provide a peak-wide threshold of significance. Peak-wide Additional replication studies. Further replication was significant SNPs identified in the GALA II study were brought for replication performed in European and African American studies from the EVE in the rest of studies. For Latinos and African Americans, we adjusted for Consortium with measured IgE levels (see Table E2 in this article’s Online global ancestry estimates, and for European Americans, we adjusted for 2 Repository at www.jacionline.org).13,26 African American studies included principal components from a principal component analysis.26 the Study of Asthma Phenotypes and Pharmacogenomic Interactions by Race-ethnicity, the Genetic Research on Asthma in the African Diaspora in Functional annotation of associated SNPs. Functional African American subjects (GRAAD-AA) and in Afro-Caribbean subjects annotation of associated SNPs and enhancer enrichment analysis was carried out with HaploReg.36 Expression quantitative trait loci (eQTLs) were from Barbados (GRAAD-AC), and the Chicago Asthma Genetics study, 37 Collaborative Studies on the Genetics of Asthma, and the Severe Asthma identified by querying the Geuvadis Data Browser. Research Program (Chicago Asthma Genetics Study/Collaborative Studies Exon sequencing. We sequenced the coding and untranslated of the Genetics of Asthma/Severe Asthma Research Program). European region exons of 3 genes, including 1 gene identified through GWASs (the American studies with measured IgE levels included the Childhood Asthma zinc finger protein 365 gene [ZNF365]) and 2 genes encoding human Management Program, the Childhood Asthma Research and Education leukocyte antigens (HLA), identified through fine mapping of admixture Network, and the Chicago Asthma Genetics Study/Collaborative Studies of mapping peaks (HLA-DQB1 and HLA-DRB5 within 6p21-22). All Puerto the Genetics of Asthma/Severe Asthma Research Program. Ricans from the GALA II study included in the study were sequenced (n 5 1454). Exons were targeted by using Molecular Inversion Probe technology, as described elsewhere.38,39 Targeted regions were sequenced on a Statistical analysis MiSeq sequencer (Illumina, San Diego, Calif) by using 150-bp reads. GWASs. Quality control was performed by removing SNPs with Output reads were aligned to the human genome (hg19) with the genotyping call rates of less than 95% and/or that deviated from Burrows-Wheeler Aligner,40 and variant calling was performed with the Hardy-Weinberg equilibrium (P <1026) within control subjects. Samples Genome Analysis Toolkit.41,42 Variants were annotated by using SeattleSeq with discrepancy between genetic sex and reported sex and with cryptic (http://snp.gs.washington.edu). relatedness (proportion of identity by descent > 0.3) were also removed. Association testing was performed by using linear regression models for Allelic association testing between genotypes at individual SNPs with natural individual variants with PLINK.43 Given that single-variant tests might have log-transformed total serum IgE levels was performed assuming an additive reduced statistical power to detect an association with rare variants, we also model by using linear regression in R 2.1427 and adjusting for age, sex, analyzed the cumulative association of multiple rare variants with IgE levels ethnicity, asthma status, and African and Native American global ancestries. by using the Sequence Kernel Association Test, as adjusted by the same We also evaluated a model adjusting for local African and Native American covariates as for individual allelic association testing.44 Sets of pooled ancestry. Regions with more than 1 SNP associated with IgE levels at a P value nonsynonymous, synonymous, and noncoding variants were analyzed of 5 3 1026 or less underwent in silico fine mapping by using genotype separately. imputation. For that, genotyped SNPs were phased with SHAPE-IT,28 A flow chart with the study design is shown in Fig E1, and additional followed by imputation with IMPUTE2,29 considering all populations from information is shown in the Methods section in this article’s Online the 1000 Genomes Project Phase I v3 as a reference.30 In addition, we Repository at www.jacionline.org. evaluated whether SNPs associated with IgE levels were associated with related phenotypes (atopic dermatitis, asthma, rhinitis, and allergic sensitization) or showed an interaction with asthma status. RESULTS Admixture mapping. Estimates of local ancestry were obtained by using the software Local Ancestry in adMixed Populations using Linkage GWASs Disequilibrium (LAMP-LD) under a 3-population model31 with reference hap- Manhattan plots of GWAS results for unadjusted models versus lotypes from the HapMap phase II CEU (European) and YRI (African) and 71 models adjusted by local genetic ancestry are shown in Figs E2, A Native American subjects genotyped on the Axiom LAT1 array.23 Inferred and B, in this article’s Online Repository at www.jacionline.org, local ancestry was then used to perform admixture mapping by correlating respectively. The quantile-quantile plot for an unadjusted model levels of local African, European, and Native American ancestry across the (see Figs E2, C) showed higher but still minimal genomic genome with natural log-transformed IgE levels by using linear regression inflation (lGC 5 1.05) than seen in the model adjusted for local and adjusting for age, sex, ethnicity, global ancestry, and asthma status. To ancestry (lGC 5 1.02; Figs E2, D). Several SNPs within a study whether overlapping peaks found for IgE levels and asthma were due 50-kb window at 10p21.2 had a P value of 5 3 1026 or less in to pleiotropic effects (ie, loci influencing IgE and asthma together) and not the model adjusted by local ancestry (see Table E3 in this article’s confounded by means of linkage disequilibrium, we used a Bayesian network method implemented in the package bnlearn for R.32 See the Methods section Online Repository at www.jacionline.org). Fine mapping of this in this article’s Online Repository at www.jacionline.org for more details. region by imputing variants from the 1000 Genomes Project The effective number of ancestry blocks was estimated by using an empiric revealed a common insertion-deletion (indel) in ZNF365 that autoregression framework with the package coda for R to account for multiple reached genome-wide significance (rs200076616, b 520.23 2 testing. On the basis of the number of ancestry blocks in the GALA II study for the insertion allele, P 5 2.3 3 10 8; Fig 1). This indel was

FIG 1. Fine mapping of ZNF365 in the GALA II study. Included in the plot are 2log10(P values) of association with total serum IgE levels by chromosome position. The most significant SNP is represented by a diamond, and the results for the remaining SNPs are color coded to show their linkage disequilibrium with this SNP based on pairwise r2 values from the Latino populations of the 1000 Genomes Project. SNPs above the dashed line are genome-wide significant (P <_ 5 3 1028). significantly associated with IgE levels in all 3 subgroups Admixture mapping of Latinos from the GALA II study: Mexicans (b 520.25, We identified 4 admixture mapping peaks whereby local 2 P 5 9.2 3 10 5), Puerto Ricans (b 520.21, P 5 8.4 3 genetic ancestry was significantly associated with IgE levels 2 10 4), and other Latinos (b 520.21, P 5 .020). However, the (P <_ 4.8 3 1025, Table I). Additional peaks showing a suggestive association was in the opposite direction in the GALA I study association are listed in Table E7 in this article’s Online (b 5 0.22, P 5 .038) and did not replicate in African Americans Repository at www.jacionline.org. Our most significant peak and European Americans from the EVE Consortium studies was located at 6p21.32-p22.1 within the MHC region (see Fig (b 520.07, P 5 .090 and b 520.06, P 5 .281, respectively). E3 in this article’s Online Repository at www.jacionline.org). 2 A summary of SNPs with a P value of 5 3 10 6 or less in models The minimum P value of the peak was located at rs3094691 in unadjusted by local ancestry is shown in Table E4 in this article’s the HLA-B gene, where Native American ancestry was associated Online Repository at www.jacionline.org. with lower total IgE levels (b 520.23, P 5 4.95 3 1028), and We next tested for replication of 23 SNPs associated with IgE European and African ancestries were associated with higher levels in prior GWASs.10-14 Fifteen of these SNPs were similarly IgE levels (b 5 0.14, P 5 4.6 3 1023 for European ancestry associated with IgE levels in the GALA II study at nominal and b 5 0.16, P 5 1.6 3 1024 for African ancestry, Fig 2). significance (P < .05): 13 were associated in the same direction The top of the peak was located 300 kb away from an admixture as the original study, 1 was associated in the opposite direction, mapping peak for asthma previously identified in the same study and the direction could not be ascertained from the original study population (GALA II study).23 A stratified analysis of asthma for the final SNP (see Table E5 in this article’s Online Repository cases and control subjects showed that the association of Native at www.jacionline.org). Nonetheless, only variants in RAD50, American ancestry with IgE at 6p21.32-p22.1 was independent HLA-DRB1, and STAT6 were significantly associated with IgE of asthma status because the effects were similar in both cases 2 levels after a Bonferroni correction (P <_ 2.2 3 10 3). However, and control subjects (b 520.23, P 5 4.8 3 1025 for cases and when looking at the gene level (65 kb), we observed a significant b 520.21, P 5 3.8 3 1024 for control subjects). We further association after Bonferroni correction at a different SNP than examined whether the association of Native American ancestry 2 initially reported for IL13 (minimum P 5 7.7 3 10 5, with IgE levels at 6p21.33 was mediating our prior association 2 rs1295686), HLA-DRB1 (minimum P 5 1.1 3 10 7 for with asthma in the same region.23 Including IgE level as a 2 rs41284732), HLA-DQB1 (minimum P 5 3.5 3 10 6 for covariate in association testing for asthma yielded a result 2 rs9273395), and IL4R/IL21R (minimum P 5 1.5 3 10 6 for that was still significant (odds ratio, 0.83; 95% CI, 0.74-0.93; rs3024667, see Table E6 in this article’s Online Repository at P 5 1.6 3 1023 vs odds ratio, 0.78; 95% CI, 0.69-0.87; www.jacionline.org). We did not find any associated SNPs within P 5 1.7 3 1025 for the adjusted and unadjusted models, or near DARC, FCER1A, HLA-G,orHLA-G after correcting for respectively). Furthermore, a Bayesian network analysis32 multiple comparisons (see Table E6). confirmed that the MHC region contains 2 independent

TABLE I. Results of admixture mapping in the GALA II study and replication in the GALA I study Discovery GALA II Replication GALA I Band Ancestry b 95% CI b P value b 95% CI b P value 6p21.32-22.1 Native 20.23 20.32 to 20.14 5.0 3 1028 20.56 20.96 to 20.16 5.7 3 1023 African 0.25 0.12 to 0.39 2.1 3 1025 0.71 0.28 to 1.14 1.3 3 1023 13p22.1-q31.3 African 20.35 20.48 to 20.21 1.0 3 1026 20.85 21.43 to 20.27 3.8 3 1023 European 0.30 0.16 to 0.43 1.8 3 1026 0.43 0.05 to 0.80 .025 14q23.2 African 0.25 0.12 to 0.39 3.0 3 1025 0.80 0.20 to 1.40 9.1 3 1023 22q13.1 African 20.69 21.01 to 20.37 1.9 3 1025 0.35 20.25 to 0.94 .252

A total of 4 significant admixture mapping peaks were identified in the GALA II study, of which 3 were significantly replicated in the same direction and with the same ancestry in the GALA I study. Significant P values after Bonferroni correction for multiple tests are shown in boldface.

FIG 2. Box plot showing the distribution of IgE levels for each copy of a chromosome of Native American (A), European (B), and African (C) ancestry at 6p21.33. associations with asthma and IgE levels, despite these admixture mapping peak (peak-wide significant, P <_ 8 3 1026; associations being correlated through linkage disequilibrium see Table E9 in this article’s Online Repository at www. (see Fig E4 in this article’s Online Repository at www. jacionline.org). Further fine mapping with imputed genotypes jacionline.org). The 3 additional admixture mapping peaks revealed 90 additional associated SNPs. A total of 39 of the 119 detected included an association between local African ancestry associated SNPs showed nominal replication in the same and higher IgE levels at 14q23.2 (b 5 0.25, P 5 3.0 3 1025) direction in the GALA I study (see Table E10 in this article’s and between local African ancestry and lower IgE levels at Online Repository at www.jacionline.org), and patterns of 13p22.1-q31.3 (b 520.35, P 5 1.0 3 1026) and 22q13.1 linkage disequilibrium were similar between the GALA I and (b 520.69, P 5 1.9 3 1025; Table I). GALA II studies within this region (see Fig E5 in this article’s Three of the 4 admixture mapping peaks were replicated in the Online Repository at www.jacionline.org). One of the imputed GALA I study after Bonferroni correction (4 peaks: P < .013): SNPs, located 21 kb 59 of HLA-DQA1, was genome-wide 6p21.32-p22.1 (P 5 5.7 3 1023 and P 5 1.3 3 1023 for Native significant in the GALA II study (rs1846190, P 5 3.5 3 1028; American and African ancestry, respectively), 13p22.1-q31.3 Fig 3) and replicated in the same direction in the GALA I study (P 5 3.8 3 1023), and 14q23.2 (P 5 9.1 3 1023, Table I). We (P 5 .003, meta-P 5 4.6 3 10210). Among the 39 replicated then brought forward the 3 peaks showing positive replication SNPs, 5 additional SNPs reached genome-wide significance in the GALA I study for replication in African Americans from when the results from the GALA II and GALA I studies were the EVE Consortium studies and used Bonferroni correction for combined (Table II). Two of these SNPs were further replicated the number of peaks tested (3 peaks: P < .017). The peaks at in European Americans (rs113176001, P 5 .010 and rs9270747, 6p21.32-p22.1 and 14q23.2 were replicated in the combined P 5 .016) but not in African Americans (see Table E10). analysis of 3187 African American subjects from the EVE The SNP rs113176001 is located 14 kb 59 of HLA-DRB1 within Consortium studies (P <_ .017, see Table E8 in this article’s histone modification marks (H3k4me1, H3k9me3, H3k04me1, Online Repository at www.jacionline.org). H3k27ac, and H3k79me2) in lymphoblastoid cells (GM12878) identified by the ENCODE project, and it is in a region that acts as a strong enhancer based on the chromatin state segmentation.45 Fine mapping of admixture mapping peaks Furthermore, rs113176001 was previously identified as a strong We next performed fine mapping of 3 admixture mapping eQTL for several proximal genes in Europeans from the 1000 217 peaks that showed significant replication in the GALA I study Genomes Project (minimum P 5 3.2 3 10 for HLA-DQA1, (6p21.32-p22.1, 13p22.1-q31.3, and 14q23.2) through allelic see Table E11 in this article’s Online Repository at www. association testing. Twenty-nine SNPs within 6p21.32-p22.1 jacionline.org) but not in subjects of African descent. were significantly associated with IgE levels after Bonferroni rs9270747 is also a strong eQTL for several proximal genes 260 correction for the number of genotyped SNPs within the (minimum P 5 1.8 3 10 for HLA-DRB5, see Table E11).

FIG 3. Fine mapping results of 6p21.32-p22.1 in the GALA II study, focusing on the region showing

genome-wide significant allelic associations. Included in the plot are 2log10(P values) of association with total serum IgE levels by chromosome position. The most significant SNP is represented by a diamond, and the results for the remaining SNPs are color coded to show their linkage disequilibrium with this SNP based on pairwise r2 values from the Latino populations of the 1000 Genomes Project. SNPs above the dashed line are genome-wide significant (P <_ 5 3 1028).

TABLE II. Summary of SNPs identified as genome-wide significantly associated with IgE levels through fine mapping of an admixture mapping peak at 6p21.32-p22.1

Gene/closest Allele GALA II study GALA I study Meta-analysis rs no. Position* gene 1/allele 2y b (95% CI) P value b (95% CI) P value b (95% CI) P value rs41284732 32551427 HLA-DRB1 T/C 20.27 1.06 3 1027 20.30 .010 20.27 3.45 3 1029 (20.37 to 20.17) (20.53 to 20.07) (20.36 to 20.18) rs186715542 32558702 39 of HLA-DRB1 C/T 0.24 (0.15 to 0.33) 4.96 3 1027 0.26 (0.02 to 0.50) .034 0.25 (0.16 to 0.33) 4.89 3 1028 rs9270747 32568292 39 of HLA-DRB1 A/G 20.19 3.42 3 1026 20.33 .002 20.21 3.56 3 1028 (20.27 to 20.11) (20.53 to 20.13) (20.29 to 20.14) rs113176001 32571483 59 of HLA-DRB1 G/C 20.29 1.03 3 1026 20.36 .008 20.30 3.04 3 1028 (20.41 to 20.17) (20.63 to 20.09) (20.41 to 20.20) rs1846190 32583813 59 of HLA-DQA1 G/A 0.22 (0.14 to 0.30) 3.51 3 1028 0.29 (0.10 to 0.48) .003 0.23 (0.16 to 0.30) 4.63 3 10210 rs557011 32587013 59 of HLA-DQA1 C/T 0.21 (0.13 to 0.29) 1.28 3 1027 0.27 (0.07 to 0.47) .007 0.21 (0.14 to 0.29) 3.55 3 1029

P values of .05 or less are shown in boldface. *According to the hg19 assembly. Allele 1 represents the allele for which effect size is reported.

Within 13p22.1-q31.3, a total of 6 SNPs were peak-wide The most significant SNP was rs2012961 (P 5 1.4 3 1024, see significantly associated with IgE levels in the GALA II study Fig E8 in this article’s Online Repository at www.jacionline. (minimum P 5 3.9 3 1026, see Tables E9 and E12 and Fig E6 in org), which is located in the 39 untranslated region of the ras this article’s Online Repository at www.jacionline.org), but none homolog family member J gene (RHOJ) and coincides with of them were replicated in the rest of studies. However, the top of the admixture mapping peak. This SNP was these SNPs showed high variability in allele frequency across replicated in the GALA I study (P 5 .019) but not in African studies/populations (see Fig E7 in this article’s Online Americans or European Americans in the EVE Consortium Repository at www.jacionline.org). Furthermore, 4 of the SNPs (Table E12). Imputation in the GALA II study revealed an had low-quality imputation scores in 1 or more studies (info additional SNP that was slightly more significant than the score < 0.4). strongest associated genotyped SNP (P 5 1.2 3 1024 for Allelic association testing of SNPs within 14q23.2 in the rs10129357 vs P 5 1.4 3 1024 for rs2012961). The imputed GALA II study did not reveal any significantly associated SNP was subsequently replicated in European Americans in the genotyped SNPs after Bonferroni correction (see Table E9). EVE Consortium (P 5 .024).

We then tested for allelic association within the admixture study alone or in a meta-analysis of the 2 studies. Two additional mapping peaks identified in the GALA II study in African admixture mapping peaks identified in the GALA II study were Americans and European Americans under the hypothesis that replicated in the GALA I study (13p22.1-q31.3 and 14q23.2), causal variants might be better tagged by different SNPs in other and one of these peaks was further replicated in African populations (see Table E13 in this article’s Online Repository at Americans (14q23.2). Fine mapping of these 2 regions revealed www.jacionline.org). In African Americans an intronic SNP allelic associations within the KCNH5-RHOJ-GPHB5 genes at within 14q23.2 was peak-wide significantly associated with IgE 14q23.2 in a combination of Latinos, European Americans, and levels after Bonferroni correction for 6511 tests (rs1741099, African Americans. b 5 0.17, P 5 3.7 3 1026). The association was replicated in We performed admixture mapping in addition to a conventional all of the non–African American samples combined (b 5 0.08, GWAS and identified additional associations with IgE levels. We P 5 .011). The SNP rs1741099 is located in intron 1 within the predicted this would be a powerful approach because of the gene encoding the glycoprotein hormone beta 5 (GPHB5; see observed differences in IgE levels among Latinos, African Fig E9 in this article’s Online Repository at www.jacionline. Americans, and European Americans16-19 and the association org) and is 23 kb away from rs2012961, the most associated between genomic African ancestry and IgE levels.20,21 Our results SNP in Latinos. We did not find any significant associations in demonstrate that local ancestry can capture additional important European Americans after adjusting for multiple tests (minimum genetic variation that is not attainable through conventional P 5 1.1 3 1024 for rs10483752 at the potassium voltage-gated GWASs. As expected, individual alleles showing an association channel, subfamily H [eag-related], member 5 [KCNH5]; see with IgE levels within the admixture mapping peaks had an Table E13). enormous variation in minor allele frequency across populations (see Fig E7). Exon sequencing further identified an association of pooled nonsynonymous variants with IgE levels in a gene Association analysis with related phenotypes implicated through admixture mapping (HLA-DQB1) but not in SNPs from STAT6 and RAD50 were nominally associated with a gene implicated through conventional GWASs (ZNF365). asthma, and SNPs from HLA-DQA1 were nominally associated Thus far, our results suggest that the association detected with with atopy and rhinitis (see Table E14 in this article’s Online local ancestry at 6p21.32-p22.1 is driven by a combination of Repository at www.jacionline.org). The SNP in GPHB5 was both common and rare variation. nominally associated with asthma, eczema, and allergic rhinitis ZNF365 encodes a protein involved in mitosis and genomic (see Table E14). One SNP from the IL4R/IL21R locus showed stability.46 Genetic variation in ZNF365 was associated with an interaction with asthma status at a P value of less than .05 atopic dermatitis in Japanese and Chinese populations47,48 but (see Table E15 in this article’s Online Repository at www. not in Europeans.49 Atopic dermatitis is an allergic disease jacionline.org). modulated through IgE, and therefore our association with IgE levels reinforces the role of ZNF365 in atopic phenotypes. However, we note that the association of the genome-wide Exon sequencing of candidate genes significant indel in ZNF365 was in the opposite direction in the Exon sequencing of HLA-DQB1, HLA-DRB5, and ZNF365 GALA I study compared with the GALA II study. Allele was performed in 1395 Puerto Ricans from the GALA II study frequencies were similar among studies, imputation performed (856 asthma cases and 539 control subjects). Gene-based well (info score >_ 0.9), and we found no significant interaction association testing revealed a significant contribution of multiple with asthma. Therefore our result could be attributed to nonsynonymous variants to IgE levels in HLA-DQB1 (Sequence differences in recruitment of patients or gene-environment Kernel Association Test, P 5 2.1 3 1023; see Fig E10 in this interactions. The associated indel is located in the first intron of article’s Online Repository at www.jacionline.org) but not in transcript variant D of ZNF365, an isoform the differential HLA-DRB5 (P 5 1.0) or ZNF365 (P 5.245). No additional single expression of which has been previously associated with Crohn variants were identified as being more strongly associated with disease.50 The combined set of variants within the first intron of IgE levels than those already identified through GWAS and ZNF365 transcript variant D with a P value of less than 1026 imputation. showed a 2.6-fold enrichment in enhancers in the lymphoblastoid cell line GM12878 (P 5.027) and a 6.7-fold enrichment in DNase sensitivity sites in CD201 B cells (P 5 .036). Therefore we DISCUSSION speculate that variation associated with IgE levels in the first In this study we performed a GWAS of IgE levels in Latinos intron of ZNF365 affects the expression, splicing, or both of and identified a novel genome-wide significant allelic association ZNF365 and might ultimately affect recombination processes at ZNF365. We also found that the majority of prior associations needed for the differentiation of B cells in IgE-producing cells, identified from GWASs primarily in European populations were as has been proposed for RAD50.51 applicable to Latino populations at the SNP level, gene-based We identified genome-wide significant associations through level, or both. We further performed the first admixture mapping admixture mapping and fine mapping in the MHC region. of IgE levels and identified a strong association between local Although this region was already known to contain genes that ancestry at the MHC region and total serum IgE levels, which influence IgE levels,10,11,13 in this study we identified an was replicated in Latinos from the GALA I study and in African important role of local genetic ancestry. In addition, our Americans from the EVE Consortium. Allelic association testing genome-wide significant associations are different from those within the MHC peak in the GALA II study revealed several SNPs previously described in GWASs involving European10,11,13 or associated with IgE levels that replicated in the GALA I study, 6 Asian14 populations. Moreover, for the first time, we implicated of which were genome-wide significant in either the GALA II variation in HLA-DQA1 in affecting IgE levels. The most

Downloaded for Anonymous User (n/a) at Henry Ford Hospital / Henry Ford Health System (CS North America) from ClinicalKey.com by Elsevier on April 21, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. J ALLERGY CLIN IMMUNOL PINO-YANES ET AL 1509 VOLUME 135, NUMBER 6 significantly associated SNP (rs113176001) is located within the help with exonic sequencing. Some computations were performed with the intergenic region between HLA-DRB1 and HLA-DQA1 and is a UCSF Biostatistics High Performance Computing System. strong eQTL for HLA-DQA1. Interestingly, this SNP was only an eQTL in lymphoblastoid cells in European but not African Key messages subjects from the 1000 Genomes Project. Furthermore, it was not significantly associated with IgE levels in African Americans d Although differences in IgE levels have been reported be- from the EVE Consortium, although the direction of effect was tween racial/ethnic groups, GWASs have predominantly the same. This could be due to variation in allele frequency focused on populations of European descent. between populations because the minor allele of this SNP is d We performed a GWAS and admixture mapping in more frequent in Latino and European populations from the Latinos and found novel associations at 3 genes/loci, 1000 Genomes Project (25% and 22% for the C allele, including ZNF365, HLA-DQA1, and 14q23.2. respectively) than in African populations (14%, see Fig E3). d Our results demonstrate how GWASs and admixture Using admixture mapping, we also uncovered an association of mapping in diverse populations can identify novel genetic African ancestry at 13p22.1-q31.3 with lower IgE levels. Fine variation that is relevant to the health of global mapping revealed significant allelic associations in the GALA II populations. study that did not replicate in the GALA I study, perhaps suggesting the presence of false-positive associations, although this might also be due to reduced statistical power from the smaller sample size in the GALA I study. African ancestry at REFERENCES 14q23.2 was associated with higher IgE levels both in Latinos and 1. Weidinger S, Baurecht H, Naumann A, Novak N. Genome-wide association African Americans. Linkage studies previously identified 14q23 studies on IgE regulation: are genetics of IgE also genetics of atopic disease? as a locus affecting IgE levels.52,53 However, no associations of Curr Opin Allergy Clin Immunol 2010;10:408-17. specific genes have been reported. Fine mapping of 14q23 in 2. Flohr C, Quinnell RJ, Britton J. Do helminth parasites protect against atopy and allergic disease? Clin Exp Allergy 2009;39:20-32. multiethnic groups narrowed the association to 3 candidate genes: 3. Naqvi M, Choudhry S, Tsai HJ, Thyne S, Navarro D, Nazario S, et al. KCNH5 and RHOJ through variants nominally associated in Association between IgE levels and asthma severity among African American, Latinos and GPHB5 in African Americans. These 3 genes are Mexican, and Puerto Rican patients with asthma. J Allergy Clin Immunol expressed by immune cells, such us T lymphocytes, B cells, 2007;120:137-43. 4. Busse W, Corren J, Lanier BQ, McAlary M, Fowler-Taylor A, Cioppa GD, et al. and plasma cells, but they have not been associated before with Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the IgE levels or allergic phenotypes. Therefore the role of genetic treatment of severe allergic asthma. J Allergy Clin Immunol 2001;108:184-90. variation in KCNH5, RHOJ, and GPHB5 on IgE levels requires 5. Lanier B, Bridges T, Kulus M, Taylor AF, Berhane I, Vidaurre CF. additional study. Omalizumab for the treatment of exacerbations in children with inadequately An important aspect of our study is that fine mapping of controlled allergic (IgE-mediated) asthma. J Allergy Clin Immunol 2009;124: 1210-6. candidate regions by using imputed genotypes from the 1000 6. Palmer LJ, Burton PR, Faux JA, James AL, Musk AW, Cookson WO. Genomes Project boosted our ability to detect novel associations. Independent inheritance of serum immunoglobulin E concentrations and airway However, a GWAS using imputed data did not reveal any responsiveness. Am J Respir Crit Care Med 2000;161:1836-43. additional associations from those already identified in our fine 7. Hopp RJ, Bewtra AK, Watt GD, Nair NM, Townley RG. Genetic analysis of allergic disease in twins. J Allergy Clin Immunol 1984;73:265-70. mapping of regions identified by GWAS/admixture mapping 8. Hanson B, McGue M, Roitman-Johnson B, Segal NL, Bouchard TJ Jr, (data not shown). One limitation of our study is that we did not Blumenthal MN. Atopic disease and immunoglobulin E in twins reared account for possible interactions between genotype and early-life apart and together. Am J Hum Genet 1991;48:873-9. exposure to allergens. In addition, IgE levels were measured by 9. Ober C, Hoffjan S. Asthma genetics 2006: the long and winding road to gene using different methods across the replication studies, which discovery. Genes Immun 2006;7:95-100. 10. Granada M, Wilk JB, Tuzova M, Strachan DP, Weidinger S, Albrecht E, et al. A might account for the lack of the replication of some of the genome-wide association study of plasma total IgE concentrations in the findings. Furthermore, we note that Latinos are a heterogeneous Framingham Heart Study. J Allergy Clin Immunol 2012;129:840-5.e21. group, and therefore findings from this study might not be 11. Moffatt MF, Gut IG, Demenais F, Strachan DP, Bouzigon E, Heath S, et al. A generalizable to all Latino populations. Finally, future studies large-scale, consortium-based genomewide association study of asthma. N Engl J Med 2010;363:1211-21. will be needed to disentangle the association at the MHC region 12. Weidinger S, Gieger C, Rodriguez E, Baurecht H, Mempel M, Klopp N, et al. by evaluating the association of classic alleles. However, better Genome-wide scan on total serum IgE levels identifies FCER1A as novel reference panels for HLA imputation need to be developed for susceptibility locus. PLoS Genet 2008;4:e1000166. Latino populations. 13. Levin AM, Mathias RA, Huang L, Roth LA, Daley D, Myers RA, et al. In summary, we confirmed associations at 6 loci previously A meta-analysis of genome-wide association studies for serum total IgE in diverse study populations. J Allergy Clin Immunol 2012;131:1176-84. associated with total serum IgE levels and identified novel 14. Yatagai Y, Sakamoto T, Masuko H, Kaneko Y, Yamada H, Iijima H, et al. associations at ZNF365, HLA-DQA1, and 14q23.2, all of which Genome-wide association study for levels of total serum IgE identifies HLA-C are likely to play an important role in total serum IgE levels in in a Japanese population. PLoS One 2013;8:e80941. Latinos. Sequencing of GWASs and admixture mapping peaks, 15. Manolio TA, Collins FS, Cox NJ, Goldstein DB, Hindorff LA, Hunter DJ, et al. Finding the missing heritability of complex diseases. Nature 2009;461:747-53. including noncoding regions, followed by functional studies is 16. Yang JJ, Burchard EG, Choudhry S, Johnson CC, Ownby DR, Favro D, et al. required to identify the causal variation behind these associations. Differences in allergic sensitization by self-reported race and genetic ancestry. J Allergy Clin Immunol 2008;122:820-7.e9. 17. Gergen PJ, Arbes SJ Jr, Calatroni A, Mitchell HE, Zeldin DC. Total IgE levels We thank the patients, families, numerous, recruiters, health care providers, and asthma prevalence in the US population: results from the National Health and community clinics for their participation. In particular, we thank Sandra and Nutrition Examination Survey 2005-2006. J Allergy Clin Immunol 2009; Salazar for her support as the GALA study coordinator and Choli Lee for his 124:447-53.

18. Litonjua AA, Celedon JC, Hausmann J, Nikolov M, Sredl D, Ryan L, et al. 36. Ward LD, Kellis M. HaploReg: a resource for exploring chromatin states, Variation in total and specific IgE: effects of ethnicity and socioeconomic status. conservation, and regulatory motif alterations within sets of genetically linked J Allergy Clin Immunol 2005;115:751-7. variants. Nucleic Acids Res 2012;40:D930-4. 19. Lester LA, Rich SS, Blumenthal MN, Togias A, Murphy S, Malveaux F, et al. 37. Lappalainen T, Sammeth M, Friedlander MR, t Hoen PA, Monlong J, Rivas MA, Ethnic differences in asthma and associated phenotypes: collaborative study on et al. Transcriptome and genome sequencing uncovers functional variation in the genetics of asthma. J Allergy Clin Immunol 2001;108:357-62. humans. Nature 2013;501:506-11. 20. Vergara C, Caraballo L, Mercado D, Jimenez S, Rojas W, Rafaels N, et al. 38. Turner EH, Lee C, Ng SB, Nickerson DA, Shendure J. Massively parallel exon African ancestry is associated with risk of asthma and high total serum IgE in capture and library-free resequencing across 16 genomes. Nat Methods 2009;6: a population from the Caribbean Coast of Colombia. Hum Genet 2009;125: 315-6. 565-79. 39. O’Roak BJ, Vives L, Fu W, Egertson JD, Stanaway IB, Phelps IG, et al. 21. Vergara C, Murray T, Rafaels N, Lewis R, Campbell M, Foster C, et al. African Multiplex targeted sequencing identifies recurrently mutated genes in autism ancestry is a risk factor for asthma and high total IgE levels in African admixed spectrum disorders. Science 2012;338:1619-22. populations. Genet Epidemiol 2013;37:393-401. 40. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler 22. Torgerson DG, Gignoux CR, Galanter JM, Drake KA, Roth LA, Eng C, et al. transform. Bioinformatics 2009;25:1754-60. Case-control admixture mapping in Latino populations enriches for known 41. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A asthma-associated genes. J Allergy Clin Immunol 2012;130:76-82.e12. framework for variation discovery and genotyping using next-generation DNA 23. Galanter JM, Gignoux CR, Torgerson DG, Roth LA, Eng C, Oh SS, et al. sequencing data. Nat Genet 2011;43:491-8. Genome-wide association study and admixture mapping identify different 42. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, et al. asthma-associated loci in Latinos: the Genes-environments & Admixture in The Genome Analysis Toolkit: a MapReduce framework for analyzing Latino Americans study. J Allergy Clin Immunol 2014;134:295-305. next-generation DNA sequencing data. Genome Res 2010;20:1297-303. 24. Reiner AP, Beleza S, Franceschini N, Auer PL, Robinson JG, Kooperberg C, et al. 43. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. Genome-wide association and population genetic analysis of C-reactive protein in PLINK: a tool set for whole-genome association and population-based linkage African American and Hispanic American women. Am J Hum Genet 2012;91: analyses. Am J Hum Genet 2007;81:559-75. 502-12. 44. Lee S, Emond MJ, Bamshad MJ, Barnes KC, Rieder MJ, Nickerson DA, et al. 25. Hoffmann TJ, Zhan Y, Kvale MN, Hesselson SE, Gollub J, Iribarren C, et al. Optimal unified approach for rare-variant association testing with application to Design and coverage of high throughput genotyping arrays optimized for small-sample case-control whole-exome sequencing studies. Am J Hum Genet individuals of East Asian, African American, and Latino race/ethnicity using 2012;91:224-37. imputation and a novel hybrid SNP selection algorithm. Genomics 2011;98: 45. Rosenbloom KR, Sloan CA, Malladi VS, Dreszer TR, Learned K, Kirkup VM, 422-30. et al. ENCODE data in the UCSC Genome Browser: year 5 update. Nucleic Acids 26. Torgerson DG, Ampleford EJ, Chiu GY, Gauderman WJ, Gignoux CR, Graves PE, Res 2013;41:D56-63. et al. Meta-analysis of genome-wide association studies of asthma in ethnically 46. Zhang Y, Park E, Kim CS, Paik JH. ZNF365 promotes stalled replication forks diverse North American populations. Nat Genet 2011;43:887-92. recovery to maintain genome stability. Cell Cycle 2013;12:2817-28. 27. R Development Core Team. R: a language and environment for statistical 47. Hirota T, Takahashi A, Kubo M, Tsunoda T, Tomita K, Sakashita M, et al. computing. Vienna: R Development Core Team; 2008. Genome-wide association study identifies eight new susceptibility loci for atopic 28. Delaneau O, Marchini J, Zagury JF. A linear complexity phasing method for dermatitis in the Japanese population. Nat Genet 2012;44:1222-6. thousands of genomes. Nat Methods 2012;9:179-81. 48. Sun LD, Xiao FL, Li Y, Zhou WM, Tang HY, Tang XF, et al. Genome-wide as- 29. Howie BN, Donnelly P, Marchini J. A flexible and accurate genotype imputation sociation study identifies two new susceptibility loci for atopic dermatitis in the method for the next generation of genome-wide association studies. PLoS Genet Chinese Han population. Nat Genet 2011;43:690-4. 2009;5:e1000529. 49. Weidinger S, Willis-Owen SA, Kamatani Y, Baurecht H, Morar N, Liang L, 30. The 1000 Genomes Project Consortium. A map of human genome variation from et al. A genome-wide association study of atopic dermatitis identifies loci population-scale sequencing. Nature 2010;467:1061-73. with overlapping effects on asthma and psoriasis. Hum Mol Genet 2013;22: 31. Baran Y, Pasaniuc B, Sankararaman S, Torgerson DG, Gignoux C, Eng C, et al. 4841-56. Fast and accurate inference of local ancestry in Latino populations. Bioinformat- 50. Haritunians T, Jones MR, McGovern DP, Shih DQ, Barrett RJ, Derkowski C, ics 2012;28:1359-67. et al. Variants in ZNF365 isoform D are associated with Crohn’s disease. Gut 32. Nagarajan R, Scutari M, Lebre S. Bayesian networks in R with applications in 2011;60:1060-7. systems biology. New York: Springer eBooks; 2013. 51. Potaczek DP, Kabesch M. Current concepts of IgE regulation and impact of 33. Shriner D, Adeyemo A, Rotimi CN. Joint ancestry and association testing in genetic determinants. Clin Exp Allergy 2012;42:852-71. admixed individuals. PLoS Comput Biol 2011;7:e1002325. 52. Mansur AH, Bishop DT, Markham AF, Morton NE, Holgate ST, Morrison 34. Kang HM, Sul JH, Service SK, Zaitlen NA, Kong SY, Freimer NB, et al. Variance JF. Suggestive evidence for genetic linkage between IgE phenotypes and component model to account for sample structure in genome-wide association chromosome 14q markers. Am J Respir Crit Care Med 1999;159: studies. Nat Genet 2010;42:348-54. 1796-802. 35. Han B, Eskin E. Random-effects model aimed at discovering associations in 53. Mansur AH, Bishop DT, Holgate ST, Markham AF, Morrison JF. Linkage/associ- meta-analysis of genome-wide association studies. Am J Hum Genet 2011;88: ation study of a locus modulating total serum IgE on chromosome 14q13-24 in 586-98. families with asthma. Thorax 2004;59:876-82.